Wang J, Barnett J T, Pollard M R, Kad N M
School of Biosciences, University of Kent, Canterbury, Kent, United Kingdom.
DFM A/S, Kongens Lyngby, Denmark.
Methods Enzymol. 2017;582:171-192. doi: 10.1016/bs.mie.2016.08.003. Epub 2016 Oct 22.
Fluorescence imaging is one of the cornerstone techniques for understanding how single molecules search for their targets on DNA. By tagging individual proteins, it is possible to track their position with high accuracy. However, to understand how proteins search for targets, it is necessary to elongate the DNA to avoid protein localization ambiguities. Such structures known as "DNA tightropes" are tremendously powerful for imaging target location; however, they lack information about how force and load affect protein behavior. The use of optically trapped microstructures offers the means to apply and measure force effects. Here we describe a system that we recently developed to enable individual proteins to be directly manipulated on DNA tightropes. Proteins bound to DNA can be conjugated with Qdot fluorophores for visualization and also directly manipulated by an optically trapped, manufactured microstructure. Together this offers a new approach to understanding the physical environment of molecules, and the combination with DNA tightropes presents opportunities to study complex biological phenomena.
荧光成像技术是理解单个分子如何在DNA上寻找其靶点的基础技术之一。通过标记单个蛋白质，可以高精度地追踪它们的位置。然而，为了理解蛋白质如何寻找靶点，有必要拉长DNA以避免蛋白质定位的模糊性。这种被称为“DNA绳索”的结构对于成像靶点位置极为强大；然而，它们缺乏关于力和负载如何影响蛋白质行为的信息。使用光镊微结构提供了施加和测量力效应的手段。在这里，我们描述了一个我们最近开发的系统，该系统能够在DNA绳索上直接操纵单个蛋白质。与DNA结合的蛋白质可以与量子点荧光团结合以进行可视化，并且还可以通过光镊制造的微结构直接操纵。这一起提供了一种理解分子物理环境的新方法，并且与DNA绳索相结合为研究复杂的生物现象提供了机会。
